Mapping Molecular Orientation with Phase Sensitive Vibrationally Resonant Sum-Frequency Generation Microscopy

Yang Han,Chao-Yu Chung,Ran-Ran Feng,Nien-Hui Ge,Hiroaki Maekawa,Varun Raghunathan,Yuan Feng,Eric O Potma

doi:10.1021/jp4022147

Abstract

We demonstrate a phase sensitive, vibrationally resonant sum-frequency generation (PSVR-SFG) microscope that combines high resolution, fast image acquisition speed, chemical selectivity, and phase sensitivity. Using the PSVR-SFG microscope, we generate amplitude and phase images of the second-order susceptibility of collagen I fibers in rat tail tendon tissue on resonance with the methylene vibrations of the protein. We find that the phase of the second-order susceptibility shows dependence on the effective polarity of the fibril bundles, revealing fibrous collagen domains of opposite orientations within the tissue. The presence of collagen microdomains in tendon tissue may have implications for the interpretation of the mechanical properties of the tissue.

Highlights

Collagen type I is the main component of tendon tissue, where the collagen molecules associate to form fibrils
A similar pattern is observed with second harmonic generation (SHG) microscopy, which is sensitive to the noncentrosymmetric arrangement of polarizable units
In order to gain further insight in the macroscopic organization of collagen in tendon tissue, we have developed and applied a new imaging technique based on sum-frequency generation (SFG)

Summary

Introduction

Collagen type I is the main component of tendon tissue, where the collagen molecules associate to form fibrils. Within the plane of the sheet, a periodic modulation of the light intensity is seen in the direction perpendicular to the long axis of fibers, suggesting an undulating arrangement of fibrils.[2] A similar pattern is observed with second harmonic generation (SHG) microscopy, which is sensitive to the noncentrosymmetric arrangement of polarizable units. In the case of collagen, the units correspond to moieties of the molecule that are arranged in a noncentrosymmetric manner, an arrangement that is preserved on a supramolecular scale.[3,4] Polarization sensitive SHG imaging has revealed that the orientation of such moieties is highly conserved along the long axis of the fibril bundle, confirming the long-range order of collagen organization within the tissue.[5,6,7]

Methods

Results

Discussion

Conclusion